title: Estimating the distance separating fluorescent protein FRET pairs.
abstract: Förster resonance energy transfer (FRET) describes a physical phenomenon widely applied in biomedical research to estimate separations between biological molecules. Routinely, genetic engineering is used to incorporate spectral variants of the green fluorescent protein (GFPs), into cellular expressed proteins. The transfer efficiency or rate of energy transfer between donor and acceptor FPs is then assayed. As appreciable FRET occurs only when donors and acceptors are in close proximity (1-10nm), the presence of FRET may indicate that the engineered proteins associate as interacting species. For a homogeneous population of FRET pairs the separations between FRET donors and acceptors can be estimated from a measured FRET efficiency if it is assumed that donors and acceptors are randomly oriented and rotate extensively during their excited state (dynamic regime). Unlike typical organic fluorophores, the rotational correlation-times of FPs are typically much longer than their fluorescence lifetime; accordingly FPs are virtually static during their excited state. Thus, estimating separations between FP FRET pairs is problematic. To overcome this obstacle, we present here a simple method for estimating separations between FPs using the experimentally measured average FRET efficiency. This approach assumes that donor and acceptor fluorophores are randomly oriented, but do not rotate during their excited state (static regime). This approach utilizes a Monte-Carlo simulation generated look-up table that allows one to estimate the separation, normalized to the Förster distance, from the average FRET efficiency. Assuming a dynamic regime overestimates the separation significantly (by 10% near 0.5 and 30% near 0.75 efficiencies) compared to assuming a static regime, which is more appropriate for estimates of separations between FPs.
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, 5625 Fishers Lane, Room TS-06F: MSC 9411, Bethesda, MD 20892-9413, USA. Electronic address: stevevog@mail.nih.gov.
authors.forename: Steven S
authors.initials: SS
authors.lastname: Vogel
authors.affiliation: Department of Physics and Astronomy, Western Kentucky University, 1906 College Heights Blvd. #11077, Bowling Green, KY 42101-1077, USA.
authors.forename: B Wieb
authors.initials: BW
authors.lastname: van der Meer
authors.affiliation: Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 10 Center Dr, Room 10D14: MSC 1855, Bethesda, MD 20892-1855, USA.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Blank
mesh_terms: D000465:Algorithms; D003198:Computer Simulation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D009010:Monte Carlo Method
chemical_list: D005456:Fluorescent Dyes
keywords: GFP; Kappa squared